Predictive Value of the Hepatic Immune Predictive Index for Patients with Primary Liver Cancer Treated with Immune Checkpoint Inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have improved survival outcomes and resulted in long-term responses in primary liver cancer in some patients. Nevertheless, not all patients with PLC could benefit from immunotherapy. Therefore, it is necessary to identify patients suitable for such therapy. METHODS: 215 patients with primary liver cancer with immunotherapy from Nanfang Hospital were screened between August 2018 and October 2020 as a training set and our validation set included 71 patients of hepatocellular carcinoma from Jiangxi Cancer Hospital from May 2019 to July 2021. The primary endpoint was the disease control rate (DCR), and the secondary endpoints were overall survival (OS) and progression-free survival. RESULTS: In the training set, neutrophil-lymphocyte ratio (NLR) ≥3 and alpha-fetoprotein (AFP) level ≥20 ng/mL were independently associated with non-DCR in the training set after adjusting for distant metastasis at baseline and targeted therapy combination. Furthermore, a hepatic immune predictive index (HIPI) based on NLR and AFP level was developed and patients with poor HIPI associated with worse clinical outcomes. In validation set, high HIPI was associated with poor OS. CONCLUSION: HIPI, based on NLR and AFP level, is an effective indicator in ICI-treated patients with primary liver cancer. Our findings may help guide the selection and on-treatment strategies for immunotherapies for primary liver cancer patients.

Identification of Prognostic Stromal-Immune Score-Based Genes in Hepatocellular Carcinoma Microenvironment.

A growing amount of evidence has suggested the clinical importance of stromal and immune cells in the liver cancer microenvironment. However, reliable prognostic signatures based on assessments of stromal and immune components have not been well-established. This study aimed to identify stromal-immune score-based potential prognostic biomarkers for hepatocellular carcinoma. Stromal and immune scores were estimated from transcriptomic profiles of a liver cancer cohort from The Cancer Genome Atlas using the ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumors using Expression data) algorithm. Least absolute shrinkage and selection operator (LASSO) algorithm was applied to select prognostic genes. Favorable overall survivals and progression-free interval were found in patients with high stromal score and immune score, and 828 differentially expressed genes were identified. Functional enrichment analysis and protein-protein interaction networks further showed that these genes mainly participated in immune response, extracellular matrix, and cell adhesion. MMP9 (matrix metallopeptidase 9) was identified as a prognostic tumor microenvironment-associated gene by using LASSO and TIMER (Tumor IMmune Estimation Resource) algorithms and was found to be positively correlated with immunosuppressive molecules and drug response.

Reciprocal regulation of HIF-1α and Uroplakin 1A promotes glycolysis and proliferation in Hepatocellular Carcinoma.

Uroplakin 1A (UPK1A) has recently been found dysregulation in many cancers. However, the functions of UPK1A and its underlying mechanisms in hepatocellular carcinoma (HCC) remain poorly understand. In the present study, we found that UPK1A was highly expressed in HCC tumor tissues compared with adjacent non-tumor tissues. Datasets from the Cancer Genome Atlas project (TCGA) and Gene expression Omnibus confirmed that UPK1A was highly expressed in HCC. High expression of UPK1A predicted poor overall survival (OS) in patients with HCC. Univariate and multivariate analysis showed that UPK1A was a significant and independent prognostic predictor for OS of patients with HCC. Functionally, silencing UPK1A suppressed HCC cell glycolysis and proliferation. Mechanistically, hypoxia-inducible factor 1α (HIF-1α) directly bound to the hypoxia response elements (HRE) of UPK1A promoter region, which led to the up-regulation of UPK1A under hypoxia. Furthermore, downregulation of UPK1A reduced key enzyme of glycolysis via regulating HIF-1α. Taken together, these data indicates the existence of a positive feedback loop between HIF-1α and UPK1A that modulates glycolysis and proliferation under hypoxia in HCC cells.

Long noncoding RNA UPK1A-AS1 indicates poor prognosis of hepatocellular carcinoma and promotes cell proliferation through interaction with EZH2.

BACKGROUND: Dysregulation of long non-coding RNAs (lncRNAs) is responsible for cancer initiation and development, positioning lncRNAs as not only biomarkers but also promising therapeutic targets for cancer treatment. A growing number of lncRNAs have been reported in hepatocellular carcinoma (HCC), but their functional and mechanistic roles remain unclear. METHODS: Gene Set Enrichment Analysis was used to investigate the molecular mechanism of UPK1A antisense RNA 1 (UPK1A-AS1). Cell Counting Kit-8 assays, EdU assays, flow cytometry, western blotting, and xenograft assays were used to confirm the role of UPK1A-AS1 in the proliferation of HCC cells in vitro and in vivo. Bioinformatics analyses and quantitative polymerase chain reaction (qRT-PCR) were performed to explore the interplay between UPK1A-AS1 and enhancer of zeste homologue 2 (EZH2). RNA immunoprecipitation (RIP), RNA pull-down assays, western blotting, and qRT-PCR were conducted to confirm the interaction between UPK1A-AS1 and EZH2. The interaction between UPK1A-AS1 and miR-138-5p was examined by luciferase reporter and RIP assays. Finally, the expression level and prognosis value of UPK1A-AS1 in HCC were analyzed using RNA sequencing data from The Cancer Genome Atlas datasets. RESULTS: We showed that UPK1A-AS1, a newly identified lncRNA, promoted cellular proliferation and tumor growth by accelerating cell cycle progression. Cell cycle-related genes, including CCND1, CDK2, CDK4, CCNB1, and CCNB2, were significantly upregulated in HCC cells overexpressing UPK1A-AS1. Furthermore, overexpression of UPK1A-AS1 could protect HCC cells from cis-platinum toxicity. Mechanistically, UPK1A-AS1 interacted with EZH2 to mediate its nuclear translocation and reinforce its binding to SUZ12, leading to increased H27K3 trimethylation. Targeting EZH2 with specific small interfering RNA impaired the UPK1A-AS1-mediated upregulation of proliferation and cell cycle progression-related genes. Moreover, miR-138-5p was identified as a direct target of UPK1A-AS1. Additionally, UPK1A-AS1 was significantly upregulated in HCC, and the upregulation of UPK1A-AS1 predicted poor prognosis for patients with HCC. CONCLUSIONS: Our study revealed that UPK1A-AS1 promotes HCC development by accelerating cell cycle progression through interaction with EZH2 and sponging of miR-138-5p, suggesting that UPK1A-AS1 possesses substantial potential as a novel biomarker for HCC prognosis and therapy.

HRG switches TNFR1-mediated cell survival to apoptosis in Hepatocellular Carcinoma.

Background: Tumor necrosis factor receptor 1 (TNFR1) signaling plays a pleiotropic role in the development of hepatocellular carcinoma (HCC). The formation of TNFR1-complex I supports cell survival while TNFR1-complex II leads to apoptosis, and the underlying mechanisms of the transformation of these TNFR1 complexes in HCC remain poorly defined. Methods: The interaction protein of TNFR1 was identified by GST pulldown assay, immunoprecipitation and mass spectrometry. In vitro and in vivo assay were performed to explore the biological features and mechanisms underlying the regulation of TNFR1 signals by histidine-rich glycoprotein (HRG). Data from the public databases and HCC samples were utilized to analyze the expression and clinical relevance of HRG. Results: HRG directly interacted with TNFR1 and stabilized TNFR1 protein by decreasing the Lys(K)-48 ubiquitination mediated-degradation. The formation of TNFR1-complex II was prompted by HRG overexpression via upregulating Lys(K)-63 ubiquitination of TNFR1. Besides, overexpression of HRG suppressed expression of pro-survival genes by impairing the activation of NF-κB signaling in the presence of TNFR1. Moreover, downregulation of HRG was a result of feedback inhibition of NF-κB activation in HCC. In line with the pro-apoptotic switch of TNFR1 signaling after HRG induction, overexpression of HRG inhibited cell proliferation and increased apoptosis in HCC. Conclusions: Our findings illustrate a crucial role for HRG in suppressing HCC via inclining TNFR1 to a pro-apoptotic cellular phenotype. Restoring HRG expression in HCC tissues might be a promising pharmacological approach to blocking tumor progression by shifting cellular fate from cell survival to apoptosis.

Nicotinamide nucleotide transhydrogenase acts as a new prognosis biomarker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Lipid metabolism is essential for cancer development. Nicotinamide nucleotide transhydrogenase (NNT) is abnormally expressed in multiple cancers; however, its role in HCC is unclear. We assessed the NNT expression level in The Cancer Genome Atlas (TCGA) cohort and Gene Expression Omnibus (GEO) datasets and found that the expression level of NNT was lower in HCC patients than non-cancer control subjects in the public databases. Survival analysis was conducted according to high and low NNT expression. Low NNT expression was significantly associated with a poor prognosis. For confirmation, the gene and protein expression of NNT in cancer and adjacent non-cancer tissues from HCC patients at our institute cohort indicated the lower expression level of NNT in cancer compared to adjacent non-cancer tissues using quantitative polymerase chain reaction and western blot, respectively. Bioinformatics was used to analyze the underlying mechanisms and establish the protein-protein interaction network of NNT. It showed that NNT is associated with functions of bile acid and fatty acid metabolism and their related genes. To conclude, our results supported that NNT expression is downregulated in HCC, and can serve as a novel prognostic biomarker.

Self-enforcing HMGB1/NF-κB/HIF-1α Feedback Loop Promotes Cisplatin Resistance in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is ranked the sixth most common cancer and the fourth leading cause of cancer-related death worldwide, and its incidence is expected to increase in the future. Cisplatin has been widely used in chemotherapy and transarterial chemoembolization in treatment for HCC. However, the main obstacle to the clinical use of cisplatin is the development of resistance, the mechanisms of which are poorly defined. Therefore, it is imperative to investigate the cellular mechanisms mediating cisplatin resistance in HCC. Here, we demonstrated that high mobility group box 1 (HMGB1) is upregulated in patients with cancer, and implicated in a tumor-supportive role. Further, we showed that HMGB1 has an important role in mediating cisplatin resistance via an HMGB1/ nuclear factor kappa-B (NF-κB)/ hypoxia inducible factor-1α (HIF-1α) feedback loop. The study findings reveal an unappreciated molecular mechanism of HMGB1-mediated cisplatin resistance and may provide a new clue in cancer therapy.

PFKFB3/HIF-1α feedback loop modulates sorafenib resistance in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a prevalent malignancy with increasing incidence and extremely poor prognosis worldwide. The multi-kinase inhibitor sorafenib is widely used as a first-line systematic treatment agent of advanced hepatocellular carcinoma. However, the benefit of sorafenib in clinical treatment is often impeded by drug resistance. Therefore, it is of critical importance to investigate the molecular mechanisms underlying sorafenib resistance in HCC. The present study shows that expression of the key glycolytic enzyme PFKFB3 is significantly up-regulated in both HCC cell lines and tissues. Thereafter, the expression of PFKFB3 was elevated in hepatocellular carcinoma cell after sorafenib treatment, which was confirmed in Gene Expression Omnibus (GEO) datasets. As predicted, the overexpression of PFKFB3 significantly enhanced HCC cells resistance to sorafenib by decreasing expression of the apoptosis-related molecules as well as apoptotic cells. Additionally, blockage of hypoxia-inducible factor-1α (HIF-1α) restricted the enhancement of PFKFB3. More interestingly, we initially found that exogenous expression of PFKFB3 significantly up-regulated the protein levels of HIF-1α in both SK-Hep-1 and SMMC-7721 cells. Further mechanistic study uncovered that HIF-1α deficiency impaired sorafenib resistance induced by PFKFB3 overexpression in HCC cells. To conclude, here we reveal a previously unrecognised positive feedback loop exists between PFKFB3 and HIF-1α and a novel HIF-1α-dependent role of PFKFB3 in regulating sorafenib resistance in HCC cells, suggesting new potential therapeutic targets for HCC treatment.

The TCP1 ring complex is associated with malignancy and poor prognosis in hepatocellular carcinoma.

TCP1 ring complex (TRiC) participates in protein folding in cells, regulating the expression of many tumor-related proteins and the cell cycle. Although the clinical significance of its subunits has been widely discussed in various malignancies, limited studies have explored its function in hepatocellular carcinoma (HCC) in the perspective of a complex. This study discusses the clinical significance of the TRiC subunits in HCC patients in terms of expression level, prognostic value, and potential mechanism. We used HCC samples from Nanfang hospital, data from The Cancer Genome Atlas (TCGA) database and information from the Gene Expression Omnibus (GEO) database with statistical methods and Gene Set Enrichment Analysis (GSEA) to analyze the gene expression levels of TRiC subunits along with survival data. We found altered expressions of the TRiC subunits in HCC, including significantly increased TCP1/CCT2/CCT3/CCT4/CCT5/CCT6A/CCT7/CCT8 expressions as well as decreased CCT6B expression, which predict poor prognosis and are associated with tumor progression. Moreover, the expression levels of these genes were pairwise correlated in HCC, indicating that the function of the entire complex should be explored as a functional macrocosm. Finally, we identified that the overexpressions of TCP1/CCT2/CCT3/CCT4/CCT5/CCT6A are involved in the dysregulation of Myc target genes, hypoxia-inducible factor (HIF) target genes and cell cycle especially the G1/S transition. Our study found that all TRiC subunits are aberrantly co-expressed in HCC, and these components have potential as therapeutic targets.

Identification and Functional Characterization of Long Non-coding RNA MIR22HG as a Tumor Suppressor for Hepatocellular Carcinoma.

Long non-coding RNAs (lncRNAs) have recently been identified as critical regulators in tumor initiation and development. However, the function of lncRNAs in human hepatocellular carcinoma (HCC) remains largely unknown. Our study was designed to explore the biological function and clinical implication of lncRNA MIR22HG in HCC. Methods: We evaluated MIR22HG expression in 52-patient, 145-patient, TCGA, and GSE14520 HCC cohorts. The effects of MIR22HG on HCC were analyzed in terms of proliferation, invasion, and metastasis, both in vitro and in vivo. The mechanism of MIR22HG action was explored through bioinformatics, luciferase reporter, and RNA immunoprecipitation analyses. Results:MIR22HG expression was significantly down-regulated in 4 independent HCC cohorts compared to that in controls. Its low expression was associated with tumor progression and poor prognosis of patients with HCC. Forced expression of MIR22HG in HCC cells significantly suppressed proliferation, invasion, and metastasis in vitro and in vivo. Mechanistically, MIR22HG derived miR-22-3p to target high mobility group box 1 (HMGB1), thereby inactivating HMGB1 downstream pathways. Additionally, MIR22HG directly interacted with HuR and regulated its subcellular localization. MIR22HG competitively bound to human antigen R (HuR), resulting in weakened expression of HuR-stabilized oncogenes, such as ß-catenin. Furthermore, miR-22-3p suppression, HuR or HMGB1 overexpression rescued the inhibitory effects caused by MIR22HG overexpression. Conclusion: Our findings revealed that MIR22HG plays a key role in tumor progression by suppressing the proliferation, invasion, and metastasis of tumor cells, suggesting its potential role as a tumor suppressor and prognostic biomarker in HCC.

Increased mediator complex subunit 15 expression is associated with poor prognosis in hepatocellular carcinoma.

Mediator complex subunit 15 (MED15) is a coactivator involved in the regulated transcription of RNA polymerase II-dependent genes and serves an oncogenic role in numerous types of cancer. However, the expression and function of MED15 in hepatocellular carcinoma (HCC) remain unknown. In the present study, the aim was to investigate the expression and clinical significance of MED15 in HCC. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical analysis revealed that MED15 mRNA and protein levels were significantly upregulated in HCC tissues compared with those in the corresponding adjacent non-tumor liver tissues. Furthermore, analyzing data from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) and GSE14520 datasets revealed a significant correlation between MED15 expression and the tumor size (P=0.033), Barcelona Clinic Liver Cancer stage (P=0.031), α-fetoprotein levels (P=0.002) and metastasis risk (P=0.001). Furthermore, patients with high MED15 expression levels had a shorter survival time compared with those with low MED15 expression levels (P<0.05). Univariate and multivariate analyses further revealed that MED15 may be an independent prognostic factor for the overall survival of HCC patients (hazard ratio, 1.762; 95% confidence interval, 1.077-2.882; P<0.05). In addition, MED15 expression was positively associated with hypoxia-inducible factor 1α expression in the TCGA-LIHC and GSE14520 datasets (P<0.01). In conclusion, the data reported in the present study indicated that MED15 is overexpressed in HCC and may represent a novel prognostic biomarker for patients with HCC.

miR-139-5p inhibits aerobic glycolysis, cell proliferation, migration, and invasion in hepatocellular carcinoma via a reciprocal regulatory interaction with ETS1.

Cancer cells have metabolic features that allow them to preferentially metabolize glucose through aerobic glycolysis, providing them with a progression advantage. However, microRNA (miRNA) regulation of aerobic glycolysis in cancer cells has not been extensively investigated. We addressed this in the present study by examining the regulation of miR-139-5p on aerobic glycolysis of hepatocellular carcinoma (HCC) using clinical specimens, HCC cells, and a mouse xenograft model. We found that overexpressing miR-139-5p restrained aerobic glycolysis, suppressing proliferation, migration, and invasion in HCC cells. miR-139-5p regulated hexokinase 1 (HK1) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression by directly targeting the transcription factor ETS1, which bound to the promoters of the HK1 and PFKFB3 genes. miR-139-5p-induced aerobic glycolysis, proliferation, migration, and invasion were reversed by ETS1 overexpression, while ETS1 silencing induced the expression of miR-139-5p via a post-transcriptional regulation mode involving Drosha. miR-139-5p expression was reduced in HCC compared to para-carcinoma tissue, which was confirmed in The Cancer Genome Atlas and GSE54751 HCC cohorts. Notably, the lower expression of mir-139 was correlated with worse prognosis. These outcomes indicate that reciprocal regulatory interactions between miR-139-5p and ETS1 modulate aerobic glycolysis, proliferation, and metastasis in HCC cells, suggesting new targets for HCC treatment.

Downregulation of Talin1 promotes hepatocellular carcinoma progression through activation of the ERK1/2 pathway.

Talin1 is an adaptor protein that conjugates integrins to the cytoskeleton and regulates integrins and focal adhesion signaling. Several studies have found that Talin1 is overexpressed in several tumor types and promotes tumor progression. However, the explicit role of Talin1 in hepatocellular carcinoma (HCC) progression is still unclear and its functional mechanism remains largely unknown. In this study, we showed a trend of gradually decreasing expression of Talin1 from normal liver tissues to hepatocirrhosis, liver hyperplasia, the corresponding adjacent non-tumor, primary HCC, and eventually metastatic foci, indicating that Talin1 may correlate with HCC initiation to progression. Talin1 was significantly downregulated in HCC tissues compared with adjacent non-tumor tissues and low Talin1 expression was associated with HCC progression and poor prognosis. Furthermore, Talin1 knockdown induced epithelial-mesenchymal transition and promoted migration and invasion in SK-Hep-1 cells and HepG2 cells. Mechanistically, we found that the ERK pathway was responsible for these promoting effects of Talin1 knockdown in HCC cells. The promoting effects of Talin1 knockdown on epithelial-mesenchymal transition, migration, and invasion were reversed by U0126, a specific ERK1/2 inhibitor. Taken together, our results suggested that Talin1 might serve as a tumor suppressor in HCC and a potential prognostic biomarker for HCC patients.

[Expression of microRNA-107 in hepatocellular carcinoma and its clinical significance].

OBJECTIVE: To investigate the expression of microRNA-107 (miR-107) and its functional role in hepatocellular carcinoma(HCC). METHODS: The gene chip data of HCC obtained from the Gene Expression Omnibus (GEO) database and the Cancer Genome Atlas (TCGA) database were used to analyze the expression levels of miR-107 in liver cancer. Twenty-two pairs of fresh surgical specimens of HCC and adjacent tissues and 53 paraffin-embedded specimens of HCC were examined for miR-107 expression by qRT-PCR. The correlation of the expression levels of miR-107 with the clinicopathologic characteristics of the patients were analyzed. The role of miR-107 in regulating the proliferation of hepatocellular carcinoma cells were determined by MTT assay in Huh7 cells transfected with a miR-107 mimic or inhibitor. RESULTS: The expression levels of miR-107 were significantly up-regulated in HCC tissues as compared to the adjacent tissues (P<0.05) in positive correlation with the tumor size (P<0.032). Transfection with miR-107 mimics significantly promoted the cell proliferation (P<0.0001) while miR-107 inhibitor inhibited the cell proliferation (P<0.0001). CONCLUSION: The expression of miR-107 is up- regulated in HCC tissues and its expression levels are correlated with HCC cell proliferation, suggesting its role as a potential oncogene in liver cancer.

Allicin sensitizes hepatocellular cancer cells to anti-tumor activity of 5-fluorouracil through ROS-mediated mitochondrial pathway.

Drug resistance and hepatic dysfunction are the two major factors that limit the application of chemotherapy for hepatocellular carcinoma (HCC). It has been reported that allicin has the hepatic protective effect and antitumor activity. Hence allicin may be an ideal enhancer to chemotherapy regimen of HCC. In the present study, we demonstrated that allicin enhanced 5-fluorouracil (5-FU) inducing cytotoxicity in HCC cells. In vivo experiment, combined treatment group with allicin (5 mg/kg/d; every two days for 3 weeks) and 5-FU (20 mg/kg/d; 5 consecutive days) showed a dramatic inhibitory effect on the growth of HCC xenograft tumors in nude mice. The co-treatment group showed highly apoptotic level compared with 5-FU treated alone. Cells combined treatment with allicin and 5-FU increased intracellular reactive oxygen species (ROS) level, reduced mitochondrial membrane potential (ΔΨm), activated caspase-3 and PARP, and down-regulated Bcl-2 compared with DMSO, allicin and 5-FU treated alone. Moreover, the increase of activated caspase-3 and PARP was blocked by the ROS inhibitor antioxidant N-acetyl cysteine (NAC). In conclusion, this is the first study to demonstrate that allicin sensitized HCC cells to 5-FU induced apoptosis through ROS-mediated mitochondrial pathway. These results provided evidences for the combination used of allicin and 5-FU as a novel chemotherapy regimen in HCC.